The present invention relates to a method for measuring an enzyme reaction and, more particularly, to a novel method for measuring the Limulus reaction or the phenol oxidase precursor cascade reaction.
It is highly necessary, especially in the medical field, to measure the enzyme reaction whose parameter changes with time. However, it is difficult to accurately measure the reaction unless the rate of change of the parameter of the enzyme reaction is constant.
For example, many infectious diseases are known in the medical field. The greatest care is given to prevent the infectious diseases because there are many cases in which patients become serious due to decrease in immunological competence. It is known that the diagnosis of the infectious diseases is possible by determining substances involved in the enzyme reaction such as endotoxin, (1.fwdarw.3)-.beta.-D-glucan, and peptidoglycan which are derived from causative bacteria.
The determination of the amount of endotoxin has conventionally been accomplished by the biological test called rabbit pyrogen test. This test involves problems of individual differences and cost, and presents difficulties in carrying out a large number of tests. Therefore, an endotoxin test using the Limulus reaction is widely used as an alternative test which is a simple and economical test.
The determination using the Limulus reaction is based on the fact that endotoxin or (1.fwdarw.3)-.beta.-D-glucan sequentially activates factors in a Limulus amebocyte lysate and causes eventual gelation or hydrolysis of a synthetic peptide substrate. The Limulus reaction is characteristic in that its reaction rate in the presence of endotoxin or (1.fwdarw.3)-.beta.-D-glucan gradually increases after a certain period of lag time and then gradually decreases after the stage of reaction at a constant rate (zeroth-order reaction), giving an S-shaped reaction curve. This necessitates careful consideration in the accurate determination of the amount of endotoxin or (1.fwdarw.3)-.beta.-D-glucan based on the Limulus reaction. Several methods have been proposed so far.
Among the methods using the Limulus reaction to determine the amount of endotoxin or (1.fwdarw.3)-.beta.-D-glucan there is, for example, a kinetic assay (such as calorimetric reaction time assay, turbidimetric reaction time assay, calorimetric reaction rate assay, and turbidimetric reaction rate assay). The calorimetric reaction time assay and the tubidimetric reaction time assay are designed to measure absorbance and turbidity, respectively, and associating a time required for the absorbance or turbidity to reach a prescribed value after the start of reaction with an extent to which the enzyme reaction has proceeded, thereby measuring the Limulus reaction. From the thus obtained value is calculated the amount of endotoxin or (1.fwdarw.3)-.beta.-D-glucan. The calorimetric reaction rate assay and the turbidimetric reaction rate assay are designed to obtain a rate at which the absorbance or turbidity changes per unit time in the Limulus reaction that takes place in a prescribed period of time and associate the change rate with an extent to which the enzyme reaction has proceeded, thereby calculating the content of endotoxin or (1.fwdarw.3)-.beta.-D-glucan.
In the past, the colorimetric and turbidimetric reaction assays have been the major kinetic methods for determining the amount of endotoxin or (1.fwdarw.3)-.beta.-D-glucan. However, there is a case where it takes a certain length of time (lag time) for the Limulus amebocyte lysate to become activated after contact with endotoxin or (1.fwdarw.3)-.beta.-D-glucan, with the lag time varying depending on the concentration of endotoxin or (1.fwdarw.3)-.beta.-D-glucan, the method of their preparation, and the origins (strains) from which they were derived. There was an instance where even those samples containing endotoxin or (1.fwdarw.3)-.beta.-D-glucan in the same amount gave greatly different values depending on the derivation and the method of preparation, from one method to another. The discrepancy in measured values among the methods employed sometimes causes serious problems (such as endotoxin shock), endangering patients, for example, when the amount of endotoxin on medical instruments or in dialysate is underestimated (because of inability to detect the endotoxin contamination exceeding the tolerance, which gives the false-negative reaction).
The reaction rate assay which has been conventionally regarded as most reliable among the methods of endotoxin determination that employ the Limulus reaction, is superior in sensitivity and capability of quantitative determination. On the other hand, it is limited in the range of determination and needs complex steps (such as dilution in the case of some samples). Therefore, it is not necessarily practical.
Peptidoglycan is a substance like endotoxin and (1.fwdarw.3)-.beta.-D-glucan, which has a biological activity undesirable for living bodies. It has a variety of biological activities to living bodies, such as pyrexic activity, lowering of liver or renal function, enhancement of endotoxin activity, and adjuvant activity to heighten the immunological function. Consequently, it should be removed from living bodies, drugs, and medical instruments as in the case of endotoxin and (1.fwdarw.3)-.beta.-D-glucan.
In the meantime, it is known that the body fluid of insects contains a cascade system which is formed of a series of enzymes called phenol oxidase precursor cascade which is involved in the melanization of the body fluid. The above-mentioned (1.fwdarw.3)-.beta.-D-glucan and peptidoglycan trigger this cascade system to eventually form melanin through the cascade reaction (Onishi, Annot. Zool. Jpn., 27, 33-39 (1954); Ashida and Onishi, Arch. Biochem. Biophys., 122, 411-416 (1967); Brunet, Insect Biochem., 10, 467-500 (1980); Ashida and Yamazaki, Molting and Metamorphosis, 239-265, Japan Sci. Press (1990); Ashida and Dohke, Insect Biochem., 10, 37-47 (1980)). A reagent to determine the total amount of (1.fwdarw.3)-.beta.-D-glucan and peptidoglycan utilizing this cascade is commercially available under the trade name of "SLP Reagent" from Wako Pure Chemical Industries, Ltd. Measurement using the reagent is the reaction time assay similar to that used to measure the Limulus reaction. Unfortunately, the phenol oxidase precursor cascade reaction also gives the S-shaped reaction curve as in the case of the Limulus reaction and hence it presents difficulties in accurate determination as in the case of determination of endotoxin and (1.fwdarw.3)-.beta.-D-glucan by the Limulus reaction.
For the reasons mentioned above, there is a demand for the development of a method for measuring an amount of endotoxin, (1.fwdarw.3)-.beta.-D-glucan, or peptidoglycan by the Limulus reaction or the phenol oxidase precursor cascade reaction, which accurately reflects the content of endotoxin, (1.fwdarw.3)-.beta.-D-glucan, or peptidoglycan and can be rapidly carried out. Thus, it is an object of the present invention to provide such a method.